Melanomas are malignant tumors characterized by early metastasis, rapid development, poor prognosis and high mortality. A highly effective and convenient method is necessary for long-term treatment of melanomas. Mitoxantrone (MTO) was topically applied for melanoma therapy using an MTO ethosome gel. Firstly, an ethosome was prepared from MTO, phospholipids, ethanol and water followed by addition of hydroxypropyl methylcellulose to obtain an ethosome gel. The ethosome was characterized. The cytotoxicity on B16 melanoma cells was evaluated on an electrical cell-substrate impedance sensing system with a novel modified chip. In vivo anti-melanoma effect of the ethosome gel was explored. Immunohistochemical and flow cytometric investigations were done. The MTO ethosomes had the size of 78nm and the zeta potential of -55mV. The ethosomes were flexible vesicles and showed much higher in vitro permeability across the rat skin than MTO aqueous solutions. The ethosomes had significant cytotoxicity and higher in vivo anti-melanoma effect than MTO solutions. The calreticulin membrane translocation of B16 cells was improved by the MTO ethosomes and the cell uptake of MTO was confirmed. The MTO ethosome gel is a promising transdermal delivery system for melanoma therapy with the advantages of non-invasion and no significant side effects.